Harvest operations for recombinant proteins

ABSTRACT

The present invention contemplates a method of producing a recombinant protein comprising (a) fermenting a prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, and (b) harvesting said recombinant protein under conditions where dO 2  levels are greater than 0%, and (c) purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm. The present invention also contemplates a method of producing a recombinant protein comprising (a) fermenting a menE gene-deleted prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, (b) harvesting said recombinant protein; and (c) purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm, and wherein the recombinant protein yield is increased by about 20% or greater.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/826,166, filed on Mar. 14, 2013, which claims benefit under 35 U.S.C.§119 to U.S. Patent Application No. 61/616,297 filed Mar. 27, 2012, theentire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to improved methods for culturing recombinantproteins in prokaryotic host cells.

BACKGROUND OF THE INVENTION

The large-scale, economic purification of proteins is required forviable biotechnology products. Generally, proteins are produced by cellculture, using either mammalian or bacterial cell lines engineered toproduce the protein of interest by insertion of a recombinant plasmidcontaining the gene for that protein. Since the cell lines used areliving organisms, they must be fed with a complex growth medium whichusually contains a mixture of salts, sugars, amino acids, vitamins,trace elements and peptones. Separation of the desired protein from themixture of compounds fed to the cells and from the by-products of thecells themselves to a purity sufficient for use as a human therapeuticposes a formidable challenge.

Recombinant therapeutic proteins are commonly produced in several hostcell lines including mammalian host cells, such as, for example, murinemyeloma NS0 and Chinese Hamster Ovary (CHO) cells (Anderson, D. C andKrummen, L. (2002) Curr. Opin. Biotech. 13: 117-123; Chu, L. andRobinson, D. K. (2001) Curr. Opin. Biotechnol. 12:180-187) and bacterialhost cells including Escherichia coli (E. coli). Each cell line hasadvantages and disadvantages in terms of productivity and thecharacteristics of the proteins produced by the cells. Escherichia colihas been most extensively used for the large-scale production oftherapeutic proteins, which do not require complex glycosylation forbioactivity. Heterologous proteins expressed by E. coli may accumulateas soluble product or insoluble aggregates. Generally, to isolate theproteins, the cells may be subjected to treatments for periplasmicextraction or be lysed to release intracellular products that areotherwise inaccessible. Advances in fermentation and cell culturetechniques have greatly increased the titers of targeted recombinantproteins.

Choices of commercial production cell lines often balance the need forhigh productivity with the ability to deliver the product qualityattributes required of a given product. Under cGMP fermentationprocedures, quality is built into the entire process ensuring thatregulatory agencies requirements are met in terms of safety, productidentity, quality and purity. However, occasionally issues arise inwhich a given product does not meet its specifications. The challenge isto develop a robust process in which to identify and isolate the issue,then mitigate the issue such that process controls can be maintainedwithin established parameter ranges, and make sure the processconsistently produces a product that meets product specifications. Thereis a need in the art for mitigating or eliminating the incidence ofproducts that do not meet specifications.

SUMMARY OF THE INVENTION

The present invention contemplates a method of producing a recombinantprotein comprising (a) fermenting a prokaryotic host cell wherein saidprokaryotic host cell has been transformed with a nucleic acid encodingsaid recombinant protein, and (b) harvesting said recombinant proteinunder conditions where dissolved oxygen (dO₂) levels are greater than0%, and (c) purifying said recombinant protein to a filtered bulk forstorage (FBS), wherein said filtered bulk does not contain detectable1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, asmeasured by an ion exchange chromatography (IEC) assay at 310 nm. In oneembodiment, in the method described above, the analytical assay is byHPLC, RP HPLC, HIC HPLC, NMR, mass spectrometry, or UV spectroscopy.

The present invention contemplates a method of producing a recombinantprotein comprising (a) fermenting a prokaryotic host cell wherein saidprokaryotic host cell has been transformed with a nucleic acid encodingsaid recombinant protein, and (b) harvesting said recombinant proteinunder conditions where dO₂ levels are greater than 0%, and (c) purifyingsaid recombinant protein to a FBS, wherein said filtered bulk does notcontain detectable DHNA-recombinant protein adduct, as measured by anIEC assay at 310 nm, wherein said recombinant protein is a recombinantpolypeptide or an isolated antibody.

The present invention contemplates a method of producing a recombinantprotein comprising (a) fermenting a prokaryotic host cell wherein saidprokaryotic host cell has been transformed with a nucleic acid encodingsaid recombinant protein, and (b) harvesting said recombinant proteinunder conditions where dO₂ levels are greater than 0%, and (c) purifyingsaid recombinant protein to a FBS, wherein said filtered bulk does notcontain detectable DHNA-recombinant protein adduct, as measured by anIEC assay at 310 nm, wherein the fermentation is scale-independent.

The present invention contemplates a method of producing a recombinantprotein comprising (a) fermenting a prokaryotic host cell wherein saidprokaryotic host cell has been transformed with a nucleic acid encodingsaid recombinant protein, and (b) harvesting said recombinant proteinunder conditions where dO₂ levels are greater than 0%, and (c) purifyingsaid recombinant protein to a FBS, wherein said filtered bulk does notcontain detectable DHNA-recombinant protein adduct, as measured by anIEC assay at 310 nm, wherein said prokaryotic host cell is Escherichiacoli (E. coli), Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus.

The present invention contemplates a method of producing a recombinantprotein comprising (a) fermenting a prokaryotic host cell wherein saidprokaryotic host cell has been transformed with a nucleic acid encodingsaid recombinant protein, and (b) harvesting said recombinant proteinunder conditions where dO₂ levels are greater than 0%, and (c) purifyingsaid recombinant protein to a FBS, wherein said filtered bulk does notcontain detectable DHNA-recombinant protein adduct, as measured by anIEC assay at 310 nm, wherein said dO₂ is maintained at levels greaterthan 0% continuously throughout the harvest operations of step (b). Inone embodiment in the method described above, the harvest operationscomprise a homogenization stage. In another embodiment, the dO₂ ismaintained at about 30% to about 75% prior to homogenization. In yetanother embodiment, the dO₂ is maintained at levels greater than 75%prior to homogenization. In still another embodiment, the dO₂ ismaintained at about 50% after homogenization. In another embodiment, thedO₂ is maintained at levels greater than 50% after homogenization. Inone embodiment, the dO₂ is maintained for a period of greater than orequal to 1.5 hours. In still another embodiment, the dO₂ is maintainedfor a period of greater than or equal to 2 hours.

The present invention contemplates a method of producing a recombinantprotein comprising (a) fermenting a prokaryotic host cell wherein saidprokaryotic host cell has been transformed with a nucleic acid encodingsaid recombinant protein, and (b) harvesting said recombinant proteinunder conditions where dO₂ levels are greater than 0%, and (c) purifyingsaid recombinant protein to a FBS, wherein said filtered bulk does notcontain detectable DHNA-recombinant protein adduct, as measured by anIEC assay at 310 nm, wherein the dO₂ is maintained with overlay orsparged air, with increased back-pressure, or with agitation (i.e.stirring). In one embodiment, the overlay air is from about 0.4 vvm toabout 0.8 vvm. In another embodiment, the overlay air is targeted at 0.6vvm. In another embodiment, the increased backpressure is between about1.0 to about 30 psi. In one embodiment, the increased backpressure istargeted at 19 psi. In still another embodiment, the agitation rate isfrom about 6 Watts/L to about 8 Watts/L. In yet another embodiment, theagitation rate is at least 6 Watts/L. In another embodiment, theagitation rate is targeted at 6 Watts/L.

In another aspect of the present invention, a method of producing arecombinant protein comprising (a) fermenting a menE gene-deletedprokaryotic host cell wherein said prokaryotic host cell has beentransformed with a nucleic acid encoding said recombinant protein, (b)harvesting said recombinant protein; and (c) purifying said recombinantprotein to a FBS, wherein said filtered bulk does not contain detectableDHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm,is contemplated. As a further embodiment to the method described above,the recombinant protein yield is increased by about 20% or greater, byabout 30% or greater, by about 40% or greater, by about 50% or greater,by about 60% or greater, as compared to the yield using a controlprokaryotic host cell.

In another aspect of the present invention, a method of producing arecombinant protein comprising (a) fermenting a menE gene-deletedprokaryotic host cell wherein said prokaryotic host cell has beentransformed with a nucleic acid encoding said recombinant protein, (b)harvesting said recombinant protein; and (c) purifying said recombinantprotein to a FBS, wherein said filtered bulk does not contain detectableDHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm,is contemplated, wherein the recombinant protein yield is increased byabout 20% or greater, by about 30% or greater, by about 40% or greater,by about 50% or greater, by about 60% or greater, as compared to theyield using a control prokaryotic host cell, wherein the fermentation isscale-independent.

In another aspect of the present invention, a method of producing arecombinant protein comprising (a) fermenting a menE gene-deletedprokaryotic host cell wherein said prokaryotic host cell has beentransformed with a nucleic acid encoding said recombinant protein, (b)harvesting said recombinant protein; and (c) purifying said recombinantprotein to a FBS, wherein said filtered bulk does not contain detectableDHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm,is contemplated, wherein the recombinant protein yield is increased byabout 20% or greater, by about 30% or greater, by about 40% or greater,by about 50% or greater, by about 60% or greater, as compared to theyield using a control prokaryotic host cell, wherein said recombinantprotein is a recombinant polypeptide or an isolated antibody.

In another aspect of the present invention, a method of producing arecombinant protein comprising (a) fermenting a menE gene-deletedprokaryotic host cell wherein said prokaryotic host cell has beentransformed with a nucleic acid encoding said recombinant protein, (b)harvesting said recombinant protein; and (c) purifying said recombinantprotein to a FBS, wherein said filtered bulk does not contain detectableDHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm,is contemplated, wherein the recombinant protein yield is increased byabout 20% or greater, by about 30% or greater, by about 40% or greater,by about 50% or greater, by about 60% or greater, as compared to theyield using a control prokaryotic host cell, wherein said prokaryotichost cell is Escherichia coli (E. coli), Enterobacter, Azotobacter,Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella,Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the COC assay results of three manufacturing runs of aproduct in which two runs, Run 2 and Run 3, did not meet the expectedresults for the COC assay. PW=purified water, C=development run control,1=Run 1, 2=Run 2, and 3=Run 3.

FIG. 2A shows the UV/vis Spectra (10 cm) for Runs 1-3—Near UV, whereRuns 1-3 are represented. New absorbance peaks were observedapproximately at 320 nm and at 460 nm which were not apparent for Run 1.FIG. 2B shows the UV/vis spectra for Run 3 minus Run 1, in which thedifference of the absorbance peaks for Runs 2 and 3 can be distinguishedfrom Run 1.

FIG. 3 shows an IEC assay monitored at 310 nm for Runs 1-3. A slightshoulder peak behind the main peak was observed for Runs 2 and 3, whilethe profile for Run 1 was comparable to the Reference Material.

FIG. 4 shows a 2D LC-MS analysis of intact Runs 1-3, monitored at 280 nmand 310 nm. An expected mass was observed for Run 1, while the expectedmass and an additional mass at 157 Da were observed for Runs 2 and 3.

FIG. 5 shows a 2D-LC MS and mass identification by tryptic peptide mapwith MS detection of a collected fraction of the brown adduct—a minorpeak from the IEC assay was collected. From the 2D LC-MS analysis, inaddition to the expected mass, a +156 Da mass was observed for thefractionated shoulder peak.

FIG. 6 shows the LC-MS-MS analysis of the novel brown adduct peakobserved at 48.8 minutes at 310 nm was determined to be T20 peptide withCys182 modified with +154.006 Da. Modified (at cysteine, +154.006 Da)and free T6 and T16 peptides were also detected by mass extraction.

FIG. 7 compares 1H-15N HSQC data of product to a synthetic peptide(NH2-IVQCR-COOH) and showed a Cys NH correlation was missing in theproduct sample.

FIG. 8 shows the proposed structure confirmed by strong nOe observedbetween the CH of Cys and the NH of arginine.

Based on the NMR data collected, the proposed structure of the brownadduct is presented in FIG. 9.

FIG. 10 shows the biosynthesis pathway in prokaryotic cells to makemenaquinones.

FIG. 11 shows a representative filtered bulk recombinant product testedfor brown adduct formation by ion exchange chromatography at 310 nm andshowed no measurable adduct formation.

FIG. 12 shows an exemplary schematic of the Hi-dO process enhancementsimplemented around the harvest operations.

FIG. 13 shows a schematic that shows the three major stages of a typicalharvest operation: post-fermentation stage, a homogenization stage, thena post-homogenization stage.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

The term “agitation rate” is mixing of the culture broth or of thehomogenate, which is typically measured as revolutions per minute (rpm).In one embodiment, agitation rate can be measured by a “power per unitvolume”. For example, at 200 rpm in a 1,000 liter fermentor, theagitation rate has a value of approximately 6 Watts/L.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments, so long as they exhibit the desired biological activity.Antibodies may be murine, human, humanized, chimeric, or derived fromother species. The term “antibody,” as used herein, also refers to afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), ECD (extracellular domain), and epitope-bindingfragments of any of the above which immunospecifically bind to cancercell antigens, viral antigens or microbial antigens, single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

“Clarity, Opalescence and Coloration (COC) Assay” is defined as usingidentical test tubes of colourless, transparent, neutral glass with aflat base and an internal diameter of 15-25 mm, compare the liquid to beexamined with a reference suspension freshly prepared as describedbelow, the depth of the layer being 40 mm. The Standard color solutionslisted in the U.S. Pharmacopeia 2012 (USP Monograph 631, Color andAchromicity) or in the European Pharmacopoeia 5.0 (EP Method 2.2.2,Degree of Coloration of Liquids) can be used for confirmation of theappropriate color assignment.

The term “1,4-dihydroxy-2-naphthoate (DHNA)” is a chemical productderived from E. coli cells. Okada Y, Tsuzuki Y, Miyazaki J, Matsuzaki K,Hokari R, Komoto S, et al. (2006) Gut 55: 681-8. DHNA is an intermediatein the menaquinone (MK), also known as vitamin K2, biosynthesis pathwayof E. coli cells. Neidhardt, F. C. (2010) Escherichia coli andSalmonella (online version: Module 3.2.2 pgs. 36-37); Inledew, W. J. &R. K. Poole (1984) The respiratory chains of Escherichia coli.Microbiological reviews. 48: 222-271; Nowicka, B. & J. Cruk (2010)Occurrence, Biosynthesis and Function of Isoprenoid Quinones. Biochimicaet Biophysica Acta 1797: 1587-1605.

The term “dissolved oxygen” (dO₂) is a relative measure of the amount ofoxygen that is dissolved or carried in a given medium. It can bemeasured with a dissolved oxygen probe such as an oxygen sensor inliquid media.

The term “ferment” or “fermenting” as used herein means the process ofculturing prokaryotic host cells that have been transformed to inducethe production of a recombinant protein of interest.

The term “filtered bulk” or “filtered bulk substance (FBS)” means therecombinant protein of interest product after harvest and purification,wherein the protein has been released from the host cell, centrifugedand/or filtered to remove any cell debris, purified over suitablechromatography columns, and subsequently concentrated by a filtrationprocess.

The term “harvested cell culture fluid”, also denoted as HCCF, meansprokaryotic or eukaryotic cell culture fluid from which the cells havebeen removed, by means including centrifugation or filtration. Cellculture is the process by which either prokaryotic or eukaryotic cellsare grown under controlled conditions. The term “cell culture” refers tothe culturing of cells derived from multicellular eukaryotes, includinganimal cells or monocellular prokaryotes, including bacteria and yeast.Eukaryotic cell cultures include mammalian cells such as Chinese HamsterOvary cells, hybridomas, and insect cells. With an appropriate cellculture vessel, secreted proteins can be obtained from anchoragedependent cells or suspension cell lines. Mammalian cell culturesinclude Chinese Hamster Ovary (CHO) cells or NS0 cells.

The term “harvest operations” or “harvesting” means, without limitation,a process comprising the lysing or homogenization, and thencentrifugation and/or filtration of a fermented prokaryotic host cellculture that has been transformed to produce a recombinant protein ofinterest, in order to begin isolating and purifying said protein ofinterest.

The term “Hi-dO” as used herein refers to an enhanced process asdescribed herein which is the maintenance of a dissolved oxygen levelgreater than 0% during harvest operations. To achieve this, the presentinvention contemplates a combination of overlay air, backpressure andagitation rate that can be used to maintain the dO₂ level at or above aset-point, i.e., above 0%, or at about 30% to about 75%, or at levelsgreater than 75%, or at about 50%, or at levels greater than 50%. Inanother embodiment, those skilled in the art could also sparge air orpure oxygen into the broth directly to achieve Hi-dO of dissolved oxygenlevels greater than 0%.

The term “homogenization” as used herein means a process of lysing orthe mechanical cell lysis of prokaryotic host cells transformed with arecombinant protein of interest in order to release said protein fromthe host cell.

The term “increased back-pressure” is used to increase the oxygentransfer rate through the culture broth. Back-pressure is typicallymeasured either in psi or bar.

“Menaquinones (MK)” are vitamin K₂ homologs and serve as electronshuttle molecules in the respiratory chain between membrane boundprotein complexes during micro-aerobic and/or anaerobic conditions. Theterm “menE” is a gene in the biosynthesis pathway to make menaquinones.

The term “microbial fermentation” means cell culture of bacteria oryeast which is genetically engineered to produce proteins and smallmolecules (e.g. secondary metabolites). Fermentation is used topropagate recombinant bacteria and yeast as well as other microorganismsand produce proteins of value. The cell productivity and growth of theseorganisms are maximized by supplying particular growth media andcontrolling and various environmental factors (such as pH, temperature,and aeration). Bacterial fermentation fluid may be derived from E. colicultures.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described for example in Clackson et al (1991) Nature,352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597.

The term “overlay air” means air blown in from the top of the fermentorwhich contains the culture broth. Typically, oxygen is supplied to afermentor by bubbling air through the liquid culture medium, oftenaccompanied by vigorous agitation to effect a fine bubble dispersion.

The term “prokaryotic host cell” as used in the present invention shouldencompass those that utilize the menaquinone biosynthesis pathway. Inone embodiment, prokaryotic host cells encompass, for example,Archaebacteria and Eubacteria, such as gram-negative or gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Inone embodiment, gram-negative cells are used. In another embodiment, E.coli cells are used as hosts for the invention (Bachmann, Cellular andMolecular Biology, vol. 2 (Washington, D.C.: American Society forMicrobiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) andderivatives thereof, including strain 33D3 having genotype W3110 ΔfhuA(ΔtonA) ptr3 lacIq lacL8 ΔompT Δ(nmpC-fepE) degP41 kan^(R) (U.S. Pat.No. 5,639,635). Of course other strains and derivatives thereof, such asE. coli 294 (ATCC 31,446), E. coli B, E. coli _(λ) 1776 (ATCC 31,537)and E. coli RV308 (ATCC 31,608) are also suitable. These examples areillustrative rather than limiting. Methods for constructing derivativesof any of the above-mentioned bacteria having defined genotypes areknown in the art and described in, for example, Bass et al. (1990)Proteins, 8: 309-314. It is, of course, necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon.

As used herein, “recombinant protein” refers generally to peptides andproteins, including antibodies. Such recombinant proteins are“heterologous,” i.e., foreign to the host cell being utilized, such as ahuman protein produced by E. coli. The polypeptide may be produced as aninsoluble aggregate or as a soluble polypeptide in the periplasmic spaceor cytoplasm.

The term “scale-independent” means the volume capacity of thefermentation process of the present invention can be accomplished usingany scale, such as, for example, from about 1 liter or greater, or about10 liters or greater, or about 100 liters or greater, or about 500liters or greater, or about 1,000 liters or greater, or about 10, 000liters or greater, or about 100,000 liters or greater.

II. Modes for Carrying Out the Invention

The present invention concerns improved methods of recombinantproduction of proteins in a prokaryotic system. The invention is basedon preventing a brown adduct formation discovered during themanufacturing of a recombinant protein which caused certain lots of theproduct to not meet specifications. As illustrated in the examplesprovided herein, the problem of the brown adduct resulted from aninconsistent redox potential during the harvest operations. It has nowbeen surprisingly discovered that the brown adduct formation can beprevented by maintaining a dissolved oxygen environment greater thanzero during the harvest operations or alternatively, by geneticallydeleting the menE gene in the prokaryotic host cell genome used torecombinantly produce the recombinant protein of interest.

Recombinant Production of Recombinant Proteins in Prokaryotic Cells

In the first step of the above processes, the heterologous nucleic acid(e.g., cDNA or genomic DNA) used to produce the recombinant protein ofinterest, is suitably inserted into a replicable vector for expressionin the bacterium under the control of a suitable promoter for bacteria.Many vectors are available for this purpose, and selection of theappropriate vector will depend mainly on the size of the nucleic acid tobe inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various componentsdepending on its function (amplification of DNA or expression of DNA)and the particular host cell with which it is compatible. The vectorcomponents for bacterial transformation may include a signal sequencefor the heterologous polypeptide and will include a signal sequence andwill also include an inducible promoter for the heterologouspolypeptide. They also generally include an origin of replication andone or more marker genes, described herein.

If the heterologous polypeptide is to be secreted, the DNA encoding theheterologous polypeptide of interest herein contains a signal sequence,such as one at the N-terminus of the mature heterologous polypeptide. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the heterologous polypeptide DNA that is inserted into thevector. The heterologous signal sequence selected should be one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For bacterial host cells that do not recognize and processthe native heterologous polypeptide signal sequence, the signal sequenceis substituted by any commonly known bacterial signal sequence.

Expression vectors contain a nucleic acid sequence that enables thevector to replicate in one or more selected host cells. Such sequencesare well known for a variety of bacteria. The origin of replication fromthe plasmid pBR322 is suitable for most gram-negative bacteria.

Expression vectors also generally contain a selection gene, also termeda selectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. One example of a selectionscheme utilizes a drug to arrest growth of a host cell. Those cells thatare successfully transformed with a heterologous gene produce a proteinconferring drug resistance and thus survive the selection regimen.

The expression vector for producing a heterologous polypeptide alsocontains an inducible promoter that is recognized by the host bacterialorganism and is operably linked to the nucleic acid encoding theheterologous polypeptide of interest. It also contains a separateinducible or low-basal-expression promoter operably linked to thenucleic acid encoding the lytic enzymes. Inducible promoters suitablefor use with bacterial hosts include the .beta.-lactamase and lactosepromoter systems (Chang et al., Nature, 275: 615 (1978); Goeddel et al.,Nature, 281: 544 (1979)), the arabinose promoter system, including thearaBAD promoter (Guzman et al., J. Bacteriol., 174: 7716-7728 (1992);Guzman et al., J. Bacteriol., 177: 4121-4130 (1995); Siegele and Hu,Proc. Natl. Acad. Sci. USA, 94: 8168-8172 (1997)), the rhamnose promoter(Haldimann et al., J. Bacteriol., 180: 1277-1286 (1998)), the alkalinephosphatase promoter, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8: 4057 (1980) and EP 36,776), the P.sub.LtetO-1 andP.sub.lac/are-1 promoters (Lutz and Bujard, Nucleic Acids Res., 25:1203-1210 (1997)), and hybrid promoters such as the tac promoter. deBoeret al., Proc. Nati. Acad. Sci. USA, 80: 21-25 (1983). However, otherknown bacterial inducible promoters and low-basal-expression promotersare suitable. Their nucleotide sequences have been published, therebyenabling a skilled worker operably to ligate them to DNA encoding theheterologous polypeptide of interest or to the nucleic acids encodingthe lytic enzymes (Siebenlist et al., Cell, 20: 269 (1980)) usinglinkers or adaptors to supply any required restriction sites. If astrong and highly leaky promoter, such as the trp promoter, is used, itis generally used only for expression of the nucleic acid encoding theheterologous polypeptide and not for lytic-enzyme-encoding nucleic acid.The tac and P_(L) promoters could be used for either, but not both. Inone embodiment, the alkaline phosphatase (phoA) promoter is used for theproduct and the arabinose (ara) promoter for the lytic enzymes.

Promoters for use in bacterial systems also generally contain aShine-Dalgarno (SD) sequence operably linked to the DNA encoding theheterologous polypeptide of interest. The promoter can be removed fromthe bacterial source DNA by restriction enzyme digestion and insertedinto the vector containing the desired DNA. The phoA promoter can beremoved from the bacterial-source DNA by restriction enzyme digestionand inserted into the vector containing the desired DNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques commonlyknown to those of skill in the art. Isolated plasmids or DNA fragmentsare cleaved, tailored, and re-ligated in the form desired to generatethe plasmids required.

Suitable prokaryotic host cells for the claimed invention include anywhich utilize the biosynthesis pathway to make menaquinones, as definedherein. Some non-limiting examples may include, for example, Escherichiacoli (E. coli), Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include Luria-Bertani (LB) brothplus necessary nutrient supplements. In certain embodiments, the mediaalso contains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene. Anynecessary supplements besides carbon, nitrogen, and inorganic phosphatesources may also be included at appropriate concentrations introducedalone or as a mixture with another supplement or medium such as acomplex nitrogen source.

For accumulation of an expressed gene product, the host cell is culturedunder conditions sufficient for accumulation of the gene product. Suchconditions include, e.g., temperature, nutrient, and cell-densityconditions that permit protein expression and accumulation by the cell.Moreover, such conditions are those under which the cell can performbasic cellular functions of transcription, translation, and passage ofproteins from one cellular compartment to another for the secretedproteins, as are known to those skilled in the art.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the typical temperature ranges from about 20°C. to about 39° C. In one embodiment, the temperature is from about 25°C. to about 37° C. In another embodiment, the temperature is at about30° C.

The pH of the culture medium may be any pH from about 5-9, dependingmainly on the host organism. For E. coli, the pH is from about 6.8 toabout 7.4, or about 7.0.

For induction, typically the cells are cultured until a certain opticaldensity is achieved, e.g., an A₅₅₀ of about 80-100, at which pointinduction is initiated (e.g., by addition of an inducer, by depletion ofa repressor, suppressor, or medium component, etc.) to induce expressionof the gene encoding the heterologous polypeptide.

After product accumulation, optionally before product recovery, thebroth lysate is incubated for a period of time sufficient to release theheterologous polypeptide contained in the cells. In an alternativeembodiment, or subsequent to the preceding, the cells present in culturemay be lysed mechanically, using any mechanical means known in the art,which may include, for example, chemical lysis or osmotic shock in orderto release said protein from the host cell.

Once lysed, the lysate or homogenate may be transferred to a hold tankwhere it can await the addition of more batches of lysate/homogenateand/or where further processing may occur, such as, for example,dilution with water, addition of buffers or flocculants, pH adjustment,or altering or maintaining the temperature of the lysate/homogenate inpreparation for subsequent recovery steps.

In a subsequent step, the heterologous polypeptide, as a soluble orinsoluble product released from the cellular matrix, is recovered fromthe lysate, or homogenate, in a manner that minimizes co-recovery ofcellular debris with the product. The recovery may be done by any means,but in one embodiment, can comprise sedimenting refractile particlescontaining the heterologous polypeptide or collecting supernatantcontaining soluble product. An example of sedimentation iscentrifugation. In this case, the recovery takes place, before expandedbed adsorption (EBA) or sedimentation, in the presence of an agent thatdisrupts the outer cell wall to increase permeability and allows moresolids to be recovered. Examples of such agents include a chelatingagent such as ethylenediaminetetraacetic acid (EDTA) or a zwitterionsuch as, for example, a dipolar ionic detergent such as ZWITTERGENT 316™detergent. In one embodiment, the recovery takes place in the presenceof EDTA.

If centrifugation is used for recovery, the relative centrifugal force(RCF) is an important factor. The RCF is adjusted to minimizeco-sedimentation of cellular debris with the refractile particlesreleased from the cell wall at lysis. The specific RCF used for thispurpose will vary with, for example, the type of product to berecovered, but is at least about 3000×g, more preferably about3500-6000×g, or about 4000-6000×g.

The duration of centrifugation will depend on several factors. Thesedimentation rate will depend upon, e.g., the size, shape, and densityof the retractile particle and the density and viscosity of the fluid.The sedimentation time for solids will depend, e.g., on thesedimentation distance and rate. It is reasonable to expect that thecontinuous disc-stack centrifuges would work well for the recovery ofthe released heterologous polypeptide aggregates or for the removal ofcellular debris at large scale, since these centrifuges can process athigh fluid velocities because of their relatively large centrifugalforce and the relatively small sedimentation distance.

The heterologous polypeptide captured in the initial recovery step maythen be further purified from the contaminating protein. In oneembodiment, the aggregated heterologous polypeptide is isolated,followed by a simultaneous solubilization and refolding of thepolypeptide, as disclosed in U.S. Pat. No. 5,288,931. Alternatively, thesoluble product is recovered by standard techniques as described below.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5th edition, PartA: Fundamentals and Techniques, Heftmann, E. (ed), Elsevier SciencePublishing Company, New York, (1992); Advanced Chromatographic andElectromigration Methods in Biosciences, Deyl, Z. (ed.), ElsevierScience BV, Amsterdam, The Netherlands, (1998); Chromatography Today,Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, NewYork, (1991); Scopes, Protein Purification Principles and Practice(1982); Sambrook, J., et al. (ed), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N. Y., 1989; or Current Protocols in Molecular Biology, Ausubel,F. M., et al. (eds), John Wiley & Sons, Inc., New York. The followingprocedures are exemplary of suitable purification procedures for thesoluble heterologous polypeptide released from the periplasm or thecytoplasm, and are well known in the art: fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation;reversed-phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; and gel filtration using, for example, SEPHADEX™ G-75.

In one aspect of the invention, the antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small-scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 20 liters in volumetric capacity.

As discussed herein, the claimed invention can be used to producerecombinant proteins, including, for example, peptides and proteins,including antibodies.

Examples of recombinant peptides and proteins that can be produced bythe method of the invention include, but are not limited to, moleculessuch as, e.g., renin, a growth hormone, including human growth hormone;bovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; α1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; thrombopoietin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnaturietic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; a serum albumin such as humanserum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; integrin; protein A or D; rheumatoid factors; a neurotrophicfactor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; cardiotrophins (cardiac hypertrophy factor) such ascardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD-3,CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-13;anti-HER-2 antibody; superoxide dismutase; T-cell receptors; surfacemembrane proteins; decay accelerating factor; viral antigen such as, forexample, a portion of the AIDS envelope; transport proteins; homingreceptors; addressins; regulatory proteins.

Antibodies produced by the claimed invention may be monoclonalantibodies that are homogeneous populations of antibodies to aparticular antigenic determinant (e.g., a cancer cell antigen, a viralantigen, a microbial antigen, a protein, a peptide, a carbohydrate, achemical, nucleic acid, or fragments thereof). A monoclonal antibody(MAb) to a target-of-interest can be prepared by using any techniqueknown in the art which provides for the production of antibody moleculesby continuous cell lines in culture. These include, but are not limitedto, the hybridoma technique originally described by Köhler and Milstein(1975) Nature 256:495-497), the human B cell hybridoma technique (Kozboret al (1983) Immunology Today 4:72), and the EBV-hybridoma technique(Cole et al (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, and IgD and any subclass thereof.The hybridoma producing the MAbs of use in this invention may becultivated in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (Teng et al (1983) Proc. Natl. Acad. Sci.U.S.A. 80:7308-7312; Kozbor et al (1983) Immunology Today 4:72-79; andOlsson et al (1982) Methods in Enzymology 92:3-16).

The antibody can also be a bispecific antibody. Bispecific antibodiesmay have a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation (WO94/04690; Suresh et al (1986) Methods in Enzymology, 121:210; Rodrigueset al (1993) J. of Immunology 151:6954-6961; Carter et al (1992)Bio/Technology 10:163-167; Carter et al (1995) J. of Hematotherapy4:463-470; Merchant et al (1998) Nature Biotechnology 16:677-681.Methods for making bispecific antibodies are known in the art (Milsteinet al (1983) Nature 305:537-539; WO 93/08829; Traunecker et al (1991)EMBO J. 10:3655-3659. Using such techniques, bispecific antibodies canbe prepared for conjugation as an antibody drug conjugate (ADC) in thetreatment or prevention of disease as defined herein.

The antibody, as defined, can be a functionally active fragment,derivative or analog of an antibody that immunospecifically binds tocancer cell antigens, viral antigens, or microbial antigens or otherantibodies bound to tumor cells or matrix. In this regard, “functionallyactive” means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative or analog is derivedrecognized. Specifically, in an exemplary embodiment the antigenicity ofthe idiotype of the immunoglobulin molecule can be enhanced by deletionof framework and CDR sequences that are C-terminal to the CDR sequencethat specifically recognizes the antigen. To determine which CDRsequences bind the antigen, synthetic peptides containing the CDRsequences can be used in binding assays with the antigen by any bindingassay method known in the art, e.g. the BIA core assay (Kabat et al,(1991) in Sequences of Proteins of Immunological Interest, FifthEdition, National Institute of Health, Bethesda, Md.; Kabat et al (1980)J. of Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)2 fragments, which contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain can beproduced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Other useful antibodies are heavy chain and lightchain dimers of antibodies, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird (1988) Science 242:423-42; Huston et al., (1988) Proc.Natl. Acad. Sci. U.S.A. 85:5879-5883; and Ward et al (1989) Nature334:544-54), or any other molecule with the same specificity as theantibody.

The antibody may be a fusion protein of an antibody, or a functionallyactive fragment thereof, for example in which the antibody is fused viaa covalent bond (e.g., a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, such as at least 10, 20 or 50 amino acid portion of theprotein) that is not the antibody. The antibody or fragment thereof maybe covalently linked to the other protein at the N-terminus of theconstant domain.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. U.S.A., 81:6851-6855). A chimeric antibodyis a molecule in which different portions are derived from differentanimal species, such as those having a variable region derived frommurine monoclonal and human immunoglobulin constant regions (U.S. Pat.Nos. 4,816,567; 4,816,397). Chimeric antibodies include “primatized”antibodies comprising variable domain antigen-binding sequences derivedfrom a non-human primate (e.g., Old World Monkey, Ape etc) and humanconstant region sequences.

Chimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, can be made using standard recombinant DNAtechniques (WO 87/02671; EP 184,187; EP 171496; EP 173494; WO 86/01533;U.S. Pat. No. 4,816,567; EP 12023; Berter et al (1988) Science 240:1041-1043; Liu et al (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 3439-3443;Liu et al (1987) J. Immunol. 139: 3521-3526; Sun et al (1987) Proc.Natl. Acad. Sci. U.S.A. 84: 214-218; Nishimura et al (1987) Cancer. Res.47: 999-1005; Wood et al (1985) Nature 314: 446-449; and Shaw et al(1988) J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229:1202-1207; Oi et al (1986) BioTechniques 4: 214; U.S. Pat. No.5,225,539; Jones et al (1986) Nature 321:552-525; Verhoeyan et al (1988)Science 239: 1534; and Beidler et al (1988) J. Immunol. 141: 4053-4060;each of which is incorporated herein by reference in its entirety.

Therapeutic monoclonal antibodies that may be produced by the methods ofthe invention include, for are not limited to, trastuzumab (HERCEPTIN®,Genentech, Inc., Carter et al (1992) Proc. Natl. Acad. Sci. U.S.A.,89:4285-4289; U.S. Pat. No. 5,725,856); anti-CD20 antibodies such aschimeric anti-CD20 “C2B8” (U.S. Pat. No. 5,736,137); rituximab(RITUXAN®), ocrelizumab, a chimeric or humanized variant of the 2H7antibody (U.S. Pat. No. 5,721,108; WO 04/056312) or tositumomab(BEXXAR®); anti-IL-8 (St John et al (1993) Chest, 103:932, and WO95/23865); antibodies targeting other interleukins, such as IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13; anti-VEGFantibodies including humanized and/or affinity matured anti-VEGFantibodies such as the humanized anti-VEGF antibody huA4.6.1 bevacizumab(AVASTIN®, Genentech, Inc., Kim et al (1992) Growth Factors 7: 53-64, WO96/30046, WO 98/45331); anti-PSCA antibodies (WO 01/40309); anti-CD40antibodies, including S2C6 and humanized variants thereof (WO 00/75348);anti-CD11a (U.S. Pat. No. 5,622,700; WO 98/23761; Steppe et al (1991)Transplant Intl. 4:3-7; Hourmant et al (1994) Transplantation58:377-380); anti-IgE (Presta et al (1993) J. Immunol. 151:2623-2632; WO95/19181); anti-CD18 (U.S. Pat. No. 5,622,700; WO 97/26912); anti-IgE,including E25, E26 and E27 (U.S. Pat. Nos. 5,714,338; 5,091,313; WO93/04173; U.S. Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO98/51793); anti-TNF-alpha antibodies including cA2 (REMICADE®), CDP571and MAK-195 (U.S. Pat. No. 5,672,347; Lorenz et al (1996) J. Immunol.156(4): 1646-1653; Dhainaut et al (1995) Crit. Care Med.23(9):1461-1469); anti-Tissue Factor (TF) (EP 0 420 937 B1); anti-humanalpha 4 beta 7 integrin (WO 98/06248); anti-EGFR, chimerized orhumanized 225 antibody (WO 96/40210); anti-CD3 antibodies such as OKT3(U.S. Pat. No. 4,515,893); anti-CD25 or anti-tac antibodies such asCHI-621 SIMULECT® and ZENAPAX® (U.S. Pat. No. 5,693,762); anti-CD4antibodies such as the cM-7412 antibody (Choy et al (1996) ArthritisRheum 39(1): 52-56); anti-CD52 antibodies such as CAMPATH-1H (Riechmannet al (1988) Nature 332: 323-337); anti-Fc receptor antibodies such asthe M22 antibody directed against Fc gamma RI as in Graziano et al(1995) J. Immunol. 155(10): 4996-5002; anti-carcinoembryonic antigen(CEA) antibodies such as hMN-14 (Sharkey et al (1995) Cancer Res. 55(23Suppl): 5935s-5945s; antibodies directed against breast epithelial cellsincluding huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al (1995) Cancer Res.55(23): 5852s-5856s; and Richman et al (1995) Cancer Res. 55(23 Supp):5916s-5920s); antibodies that bind to colon carcinoma cells such as C242(Litton et al (1996) Eur J. Immunol. 26(1):1-9); anti-CD38 antibodies,e.g. AT 13/5 (Ellis et al (1995) J. Immunol. 155(2): 925-937); anti-CD33antibodies such as Hu M195 (Jurcic et al (1995) Cancer Res 55(23 Suppl):5908s-5910s and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 orLymphoCide (Juweid et al (1995) Cancer Res 55(23 Suppl): 5899s-5907s);anti-EpCAM antibodies such as 17-1A (PANOREX®); anti-GpIIb/IIIaantibodies such as abciximab or c7E3 Fab (REOPRO®); anti-RSV antibodiessuch as MEDI-493 (SYNAGIS®); anti-CMV antibodies such as PROTOVIR®);anti-HIV antibodies such as PRO542; anti-hepatitis antibodies such asthe anti-Hep B antibody OSTAVIR®); anti-CA 125 antibody OvaRex;anti-idiotypic GD3 epitope antibody BEC2; anti-human renal cellcarcinoma antibody such as ch-G250; ING-1; anti-human 17-1A antibody(3622W94); anti-human colorectal tumor antibody (A33); anti-humanmelanoma antibody R24 directed against GD3 ganglioside; anti-humansquamous-cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA)antibodies such as Smart ID10 and the anti-HLA DR antibody Oncolym(Lym-1).

II. Methods and Assays Analytical Methods/Assays Clarity, Opalescenceand Coloration (COC) Assay

The degree of opalescence may also be determined by instrumentalmeasurement of the light absorbed or scattered on account ofsubmicroscopic optical density in homogeneities of opalescent solutionsand suspensions. Such techniques are nephelometry and turbidimetry. Forturbidity measurement of coloured samples, ratio turbidimetry andnephelometry with ratio selection are used. The light scattering effectof suspended particles can be measured by observation of either thetransmitted light (turbidimetry) or the scattered light (nephelometry).Ratio turbidimetry combines the principles of both nephelometry andturbidimetry. Turbidimetry and nephelometry are useful for themeasurement of slightly opalescent suspensions. Reference suspensionsproduced under well-defined conditions must be used. Standard colorsolutions listed in the U.S. Pharmacopeia 2012 (USP Monograph 631, Colorand Achromicity) or in the European Pharmacopoeia 5.0 (EP Method 2.2.2,Degree of Coloration of Liquids) for confirmation of the appropriatecolor assignment. For quantitative measurements the construction ofcalibration curves is essential, since the relationship between theoptical properties of the suspension and the concentration of thedispersed phase is at best semi-empirical. The determination ofopalescence of coloured liquids is done with ratio turbidimeters ornephelometers with ratio selection since colour provides a negativeinterference, attenuating both incident and scattered light and loweringthe turbidity value. The effect is so great for even moderately colouredsamples that conventional nephelometers cannot be used. The instrumentalassessment of clarity and opalescence provides a more discriminatorytest that does not depend on the visual acuity of the analyst. Numericalresults are more useful for quality monitoring and process control,especially in stability studies. For example, previous numerical data onstability can be projected to determine whether a given batch of dosageformulation or active pharmaceutical ingredient will exceed shelf-lifelimits prior to the expiry date.

HPLC Assay

High Performance Liquid Chromatography, also known as High PressureLiquid Chromatography, abbreviated as HPLC, is a special form of liquidchromatography and nowadays used frequently in biochemistry andanalytical chemistry. The analyte is forced through a column of thestationary phase in a liquid (mobile phase) at high pressure, whichdecreases the time the separated components remain on the stationaryphase and thus the time they have to diffuse within the column. Thisleads to narrower peaks in the resulting chromatogram and thence tobetter resolution and sensitivity as compared to LC. The mobile phase ischosen to ensure solubility of the sample solutes. For the stationaryphase, preferably microparticulate silica (bare or chemically modified)is used, because its high surface area accentuates the differences insolute-stationary phase interactions. The use of a stationary phase thatinteracts strongly with solutes relative to solute mobile-phaseinteractions will result in very long retention times, a situation whichis not analytically useful. Hence the stationary phase must be selectedso as to provide weak to moderate solute interactions relative to thosein the mobile phase. As a consequence, the nature of the solute governsthe type of LC selected. The stronger interactions should occur in themobile phase to ensure sample solubility and ready elution, while thestationary phase should be responsive to more subtle differences amongthe solutes. For example, polar neutral compounds are usually betteranalyzed using a polar mobile phase together with a nonpolar stationaryphase that distinguishes subtle differences in the dispersive characterof the solutes. One of the powerful aspects of HPLC is that the mobilephase can be varied to alter the retention mechanism. Modifiers can beadded to the mobile phase to control retention. For example, pH is animportant variable in aqueous mobile phases.

Reversed-phase chromatography (RP-HPLC) calls for the use of a non-polarstationary phase and a polar mobile phase (composed of one or more ofthe polar solvents, e.g. water, methanol, acetonitrile, andtetrahydrofuran).

Hydrophobic interaction chromatography (HIC) HPLC: This chromatographicmethod is good for analyzing proteins or antibody/protein bioconjugatesbased on their hydrophobicity. The theory behind hydrophobic interactionchromatography is that proteins are bound to the resin by employing anaqueous high salt mobile phase. The salt conditions contribute to alyotropic effect which allows the proteins to bind to the lower surfacecoverage of a hydrophobic ligand. Proteins are eluted by the simpletechnique of decreasing the salt concentration. Most therapeutic targetsare eluted in a low salt or a no salt buffer. Thus, the compound can beeluted in a more polar and less denaturing environment. For example, HIChas been used extensively to analyze drug loading in antibody-drug orprotein-drug conjugates.

NMR Assay

Nuclear magnetic resonance (NMR) detection is based on the fact thatcertain nuclei with odd-numbered masses, including H and 13C, spin aboutan axis in, a random fashion. However, when placed between poles of astrong magnet, the spins are aligned either parallel or anti-parallel tothe magnetic field, with the parallel orientation favored since it isslightly lower in energy. The nuclei are then irradiated withelectromagnetic radiation which is absorbed and places the parallelnuclei into a higher energy state; consequently, they are now in“resonance” with the radiation. Each H or C will produce differentspectra depending on their location and adjacent molecules, or elementsin the compound, because all nuclei in molecules are surrounded byelectron clouds which change the encompassing magnetic field and therebyalter the absorption frequency.

Mass Spectrometry

Mass spectrometry is an analytical technique used to measure themass-to-charge ratio (m/z or m/q) of ions. It is most generally used toanalyze the composition of a physical sample by generating a massspectrum representing the masses of sample components. The technique hasseveral applications including identifying unknown compounds by the massof the compound and/or fragments thereof determining the isotopiccomposition of one or more elements in a compound, determining thestructure of compounds by observing the fragmentation of the compound,quantitating the amount of a compound in a sample using carefullydesigned methods (mass spectrometry is not inherently quantitative),studying the fundamentals of gas phase ion chemistry (the chemistry ofions and neutrals in vacuum), and determining other physical, chemicalor even biological properties of compounds with a variety of otherapproaches.

A mass spectrometer is a device used for mass spectrometry, and itproduces a mass spectrum of a sample to analyze its composition. This isnormally achieved by ionizing the sample and separating ions ofdiffering masses and recording their relative abundance by measuringintensities of ion flux. A typical mass spectrometer comprises threeparts: an ion source, a mass analyzer, and a detector.

The kind of ion source is a contributing factor that stronglyinfluences-what types of samples can be analyzed by mass spectrometry.Electron ionization and chemical ionization are used for gases andvapors. In chemical ionization sources, the analyte is ionized bychemical ion-molecule reactions during collisions in the source. Twotechniques often used with liquid and solid biological samples includeelectrospray ionization (ESI) and matrix-assisted laserdesorption/ionization (MALDI). Other techniques include fast atombombardment (FAB), thermospray, atmospheric pressure chemical ionization(APCI), secondary ion mass spectrometry (SIMS), and thermal ionisation.

UV Spectroscopy

Ultraviolet-visible spectroscopy or ultraviolet-visiblespectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopyor reflectance spectroscopy in the ultraviolet-visible spectral region.This means it uses light in the visible and adjacent (near-UV andnear-infrared (NIR)) ranges. The absorption or reflectance in thevisible range directly affects the perceived color of the chemicalsinvolved. In this region of the electromagnetic spectrum, moleculesundergo electronic transitions. This technique is complementary tofluorescence spectroscopy, in that fluorescence deals with transitionsfrom the excited state to the ground state, while absorption measurestransitions from the ground state to the excited state. A UVspectrometer is an instrument that uses a beam of light from a visibleand/or UV light source (colored red) is separated into its componentwavelengths by a prism or diffraction grating. Each monochromatic(single wavelength) beam in turn is split into two equal intensity beamsby a half-mirrored device. One beam, the sample beam (colored magenta),passes through a small transparent container (cuvette) containing asolution of the compound being studied in a transparent solvent. Theother beam, the reference (colored blue), passes through an identicalcuvette containing only the solvent. The intensities of these lightbeams are then measured by electronic detectors and compared. Theintensity of the reference beam, which should have suffered little or nolight absorption, is defined as I0. The intensity of the sample beam isdefined as I. Over a short period of time, the spectrometerautomatically scans all the component wavelengths in the mannerdescribed. The ultraviolet (UV) region scanned is normally from 200 to400 nm, and the visible portion is from 400 to 800 nm.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Adduct Detection

During a manufacture for a particular recombinant protein, sevenfiltered bulks for storage (FBS) were produced where typical resultsagainst product appearance criteria were obtained for five of the sevenbulks. Per manufacturing specification, the product specific testinstructions require the use of Yellow (Y) color series for theevaluation of the product samples by the COC assay, a method for thedetermination of clarity/degree of opalescence, degree of coloration,and appearance. However, two bulks (Runs 2 and 3) appeared brown incolor and did not meet the expected Yellow series color criterion of ≦Y7for the COC assay. A comparison of the COC results for Runs 1-3 is shownin FIG. 1. To investigate the discrepancy further, the seven FBS sampleswere concentrated to increase the intensity of the color. Theconcentrated samples were compared against all the Standard colorsolutions listed in the U.S. Pharmacopeia 2012 (USP Monograph 631, Colorand Achromicity) or in the European Pharmacopoeia 5.0 (EP Method 2.2.2,Degree of Coloration of Liquids) for confirmation of the appropriatecolor assignment. The samples were compared in diffused daylight 5 minafter preparation of the reference sample, viewing vertically against ablack background. The diffusion of light must be such that referencesample I can readily be distinguished from water and that referencesuspension II can readily be distinguished from reference suspension I.A liquid was considered clear if its clarity was the same as that ofwater R or of the solvent used when examined under the conditionsdescribed above, or if its opalescence was not more pronounced than thatof the reference sample I.

Since the cause of the coloration was unknown for Runs 2 and 3, multipleinvestigational studies were completed to determine the source and causeof the atypical brown color. Samples from Runs 1-3 were analyzed formetals, trace elements (other than metals), and chromophores. Thesestudies suggested that the coloration observed in Runs 2 and 3 were notdue to metals or other trace elements (data not shown).

To determine whether chromophores were associated with the unexpectedcolor observed in the FBS, Runs 1-3 were analyzed using ultraviolet andvisible (UV/vis) spectroscopy with a 1 cm path length cuvette. The UVspectra (200-600 nm) did not display any significant differences in theobservance profile for the samples analyzed.

To increase the sensitivity of the UV spectrophotometer, the experimentwas repeated using a 10 cm path length cuvette. The 10 cm cuvette offersincreased sensitivity to the 1 cm cuvette due to the absorbance of asample is proportional to the number of absorbing molecules in thespectrophotometer meter light beam. The samples were scanned between200-700 nm to determine the absorption spectrum of Runs 1-3. The shapeof the spectra for Runs 2 and 3 was different than Run 1: new absorbancepeaks were observed approximately at 320 nm and at 460 nm which were notapparent for Run 1 (FIG. 2A). This difference can be observed moreclearly when the spectrum of Run 1 is subtracted from the spectrum ofRun 3 (FIG. 2B). The peak observed at 460 nm for Runs 2 and 3 isconsistent with a flavin (e.g., vitamin) fingerprint.

Based on the 10 cm UV/vis results, full spectrum analysis for RP-HPLCand IEC with options for MS detection were performed on FBS from Runs1-3.

Using full spectrum detection for RP-HPLC, no chromatographicdifferences were observed for Run 1-3 (data not shown). However, for IECat 310 nm, minor differences were observed. As shown in FIG. 3, a slightpeak behind the Main Peak is observed for Runs 2 and 3 while the profilefor Run 1 is comparable to the Reference Material.

Intact samples were submitted for 2D LC-MS and monitored at both 280 and310 nm. The 2D LC-MS analysis consists of two parts—first dimension isseparation by RP-HPLC with the second dimension as fractionated peaksfor mass spectrometry analysis. From this experiment, the expected masswas observed for Run 1 while the expected mass and an additional mass ofapproximately +157 Da were observed for Runs 2 and 3 (FIG. 4).

Example 2 Elucidation of the Adduct

To better elucidate the adduct, Run 3 was selected for fractionation(the minor peak from the IEC assay (FIG. 3) was collected) and furtheranalyzed by 2D-LC MS and mass identification by tryptic peptide map withMS detection.

From the 2D LC-MS analysis (FIG. 5), in addition to the expected mass,an approximate +156 Da mass increase was again observed for thefractionated shoulder peak. Upon on-line reduction (with DTT) of thesample, the expected reduced mass was observed. The four additionalDaltons observed between the reduced and native analyses are due to thebreakage of the disulfide bonds and the addition of four hydrogens. Theadditional mass was again observed, suggesting the modification wasnon-reversible or covalent.

From the tryptic peptide map, the sample was collected at both 214 nmand 310 nm. As shown in FIG. 6, novel peaks are enhanced in the 45-55minute region. LC-MS-MS analysis of the novel peak observed at 48.8minutes at 310 nm was determined to be T20 peptide with the cysteineresidue modified with +154.006 Da. Modified (at cysteine, +154.006 Da)and free T6 and T16 peptides were also detected by mass extraction.Reduced T21 or modified T21 were not detected but this may have been dueto the low levels present. The other two peaks observed eluting between50 to 56 minutes at 310 nm did not contain any unique species whencompared to the reference.

1D and 2D 1H NMR analysis was collected to determine the adductstructure. Additional data was acquired using TOSCY (Total CorrelationSpectroscopy), HSQC (Heteronuclear Single Quantum Coherence), HMBC(Heteronuclear Multiple Bond Correlation), and ROESY (Rotating-frameOverhauser Effect Spectroscopy (nOe)).

TOCSY creates correlations between all protons that are coupled to eachother as well as all other protons within a given spin system. HSQCexperiment correlates chemical shifts of directly bound nuclei (i.e. twotypes of chemical nuclei) while HMBC experiment correlates chemicalshifts of two types of nuclei separated from each other with two or morechemical bonds. ROESY utilizes nOe which uses space, not throughchemical bonds to confirm a precise molecular conformation (i.e., threedimensional structure of a molecule). The collected peptide observedlong range 1H-13C coupling between aromatic (quinone) protons and C═O at182 ppm. The 1H-13C HSQC chemical shifts for the collected peptide inthe aromatic region are a close match to those observed for thesynthetic model compound bound to naphthalene-1,4-dione. TOCSY dataassigns the Q, V, and R resonances in the product. Comparing 1H-15N HSQCdata of product to a synthetic peptide (NH₂-IVOCR-COOH) showed a Cys NHcorrelation was missing in the product sample as shown in FIG. 7. Theproposed structure is confirmed by strong nOe observed between the CH ofCys and the NH of Arg (FIG. 8). Based on the NMR data collected, theproposed structure is presented in FIG. 9.

The identification of the colored species as 1,4-dihydroxy-2-naphthoate(DHNA) which formed the recombinant protein-brown adduct was based uponMS, NMR and genetic data. NMR data confirmed that DHNA was attached tothe recombinant protein via cysteine residues. DHNA is a product derivedfrom the menaquinone biosynthesis pathway of E. coli cells (FIG. 10).Menaquinone is present in E. coli but production of it is increased whenthe culture is in an anaerobic and/or micro-aerobic condition.Menaquinone is used for electron transport in limited oxygenenvironments and used for returning the disulfide bond forming proteinDsbB to the active oxidized state in anaerobic (micro-aerobic)conditions.

Example 3 Hi-dO Process to Mitigate Formation of DHNA-product Adduct

A control strategy was developed to prevent the generation of aproduct's free thiols and the subsequent formation of the DHNA-productadduct. The cause of the color formation was determined to be the resultof a low redox environment during the harvest operations because Runs 2and 3 exhibited the highest titers and cell densities, both weresubjected to longer hold times for their diluted homogenates, enduredlonger durations for the homogenates to achieve less than the 15° C.target temperature and had suboptimal homogenate mixing times and rates(data not shown). These factors contributed to generating a low oxygenenvironment which promoted the reduction of the product disulfide bondsand permitted the opportunity for DHNA to attach to the free thiols ofthe protein product.

Since the DHNA-protein adduct was formed during the low redoxenvironment during the harvest operations which led to reduced disulfidebonds (i.e. free thiols), an approach was developed to prevent thegeneration of free thiols and the formation of the DHNA-product adduct.This enhanced process control, called Hi-dO, maintains the dissolvedoxygen levels in the harvest operations at greater than zero (>0%) toeliminate the reducing environment (i.e. no free thiol generation).

The formation of the DHNA-product adduct is a complex biologicalreaction that requires the combination of multiple events across thefermentation and harvest operations. The output of the fermentationprocess is the production of considerable levels and/or availability ofDHNA. The schematic of the three major stages of a typical harvestoperation is shown on FIG. 13.

Several process steps were tested post-fermentation/pre-homogenizationand tested post-homogenization, to determine if such actions wouldmitigate the reducing environment or free thiol generation. Such processenhancements tested are shown in Table 1 and FIG. 12.

TABLE 1 Process Enhancements (Hi-dO)Post-Fermentation/Pre-Homogenization In HMG HoldTank/Post-Homogenization Initiate WCB Hi-dO process control: Dilutehomogenate with 2x water prior to homogenate 1. Target dO₂ > 75%transfer 2. Increase agitation rate (6.3 1. Temperature control to 10°C. Watts/L) 2. Target dO₂ > 50% by increasing agitation and/or air 3.Apply overlay air (0.6 vvm) sparging 4. Back-pressure added to about18.85 psi (1.3 bar) 5. Process time = 1.5 hours Transfer homogenate inwater for immediate dilution Initiate Hi-dO₂ homogenate process control:6. Maintain Target dO₂ > 50% 7. Increase agitation (1-6 Watts/L) 8.Apply overlay or sparged air (if required) 9. Process time = 2 hours

The results of the process enhancements outlined in Table 1 and FIG. 12are shown in Table 2.

TABLE 2 Product Quality Analyses of Development Runs Performed with theHi-dO Enhanced Process Controls IEC % Anomalous IEC Peak % MainFermentation @ 280 / Peak RP-HPLC SEC Native SEC Run 310 nm @ 280 % PeakA % Monomer % Monomer Small-scale 0.00/0.00 99.48 98.82 100.00 99.99 (10L) #1 Small-scale 0.00/0.00 99.69 99.00 100.00 99.97 (10 L) #2Manufacturing- 0.00/0.00 99.58 99.03 100.00 99.99 scale (1,000 L)Release Spec Not defined, ≧97% Main ≧97% Peak ≧98% ≧98% FBS CofA butshould Peak A monomer monomer not be detectable

A root cause analysis was carried out to understand the origins of thebrown coloration. This analysis resulted in the identification of thecolored species (DHNA), its attachment to a recombinant protein product,the adduct (DHNA-protein) structure, its origination and the proposedmechanism of how and when DHNA became attached to the product during theproduction process. As Table 1 summarizes, a mitigation strategy wasimplemented to prevent formation of the brown adduct, by maintaining thedissolved oxygen level greater than zero (>0%) throughout the harvestoperations to eliminate the reducing environment and prevent theformation of product free thiols. As a result, as shown by IEC analysesas the % anomalous peak demonstrated 0%, the brown adduct formation wasnot detected in the FBS (Table 2).

Example 4 Generating menE Gene-Deleted E. coli Host Cells

In addition to the Hi-dO harvest process of the invention, anotherapproach was undertaken to mitigate the brown adduct formation. Thisinvolved genetically engineering the prokaryotic host cell such that themenE gene was deleted from the genome, thereby preventing the productionof any DHNA intermediate from the menaquinone biosynthesis pathway thatcould be attached to the recombinant product.

The menE gene deleted host cells were generated as an in-frame,single-gene knockout mutant following the methods described in Baba etal., Construction of E. coli K-12 in-frame, single-gene knockoutmutants: the Keio collection, Molecular Systems Biology, vol. 21, p.1-10 (2006) which is hereby incorporated by reference. The menE gene wastargeted for mutagenesis with PCR products containing a resistancecassette (such as kanamycin) flanked by FLP recognition target sites anda 50 base pair homologies to the adjacent chromosomal sequences.

The mutagenesis yielded approximately 10-1000 kanamycin resistancecolonies when the host cells were incubated aerobically at 37° C. onLuria-Bertani broth (LB) agar containing 30 μg/mL kanamycin.

Example 5 Production of Recombinant Proteins Using menE Gene-Deleted E.coli Host Cells

The ability of the menE gene-deleted E. coli host cells to producerecombinant protein that did not exhibit DHNA-associated protein adductwas tested. Briefly, the menE gene-deleted E. coli cells weretransformed with plasmid constructs that encoded for two recombinantproteins, PROT 1 and PROT 2, and two recombinant antibodies, AB 1 andAB2, per standard techniques well-known to those of skill in the art(see for example, Simmons et al., Expression of full-lengthimmunoglobulins in E. coli: rapid and efficient production ofaglycosylated antibodies, J of Immunol Methods 263 p. 133-147 (2002)).Fermentation of the four recombinant proteins/antibodies proceeded asdescribed herein (see also U.S. Pat. No. 6,979,556 which is herebyincorporated by reference).

The filtered bulk recombinant product for all four recombinantprotein/antibodies were tested for DHNA-protein adduct formation by IECassay at 310 nm and showed no detectable DHNA-protein adduct formation(see FIG. 11 for exemplary results for PROT 1).

Surprisingly, it was found that the yield of recombinant product as aresult of using the menE deleted E. coli cells increased appreciably byabout 20% to 50% as compared to the yield using E. coli host cells withan intact, wild-type menE gene. Table 3 shows these results.

TABLE 3 Recombinant Protein Yields using menE gene-deleted host cellsYield using Yield using menE Recombinant wild-type gene-deleted %Protein E. coli host E. coli host change PROT 1 1.9 g/L 2.5 g/L 30% PROT2 5.5 g/L 6.5 g/L 20% AB1 0.7 g/L 1.0 g/L 40% AB2 0.46 g/L  0.72 g/L 50%

1. A method of producing a recombinant protein comprising (a) fermentinga prokaryotic host cell wherein said prokaryotic host cell has beentransformed with a nucleic acid encoding said recombinant protein, and(b) harvesting said recombinant protein under conditions where dissolvedoxygen (dO₂) levels are greater than 0%, and (c) purifying saidrecombinant protein to a filtered bulk for storage (FBS), wherein saidfiltered bulk does not contain detectable 1,4-dihydroxy-2-naphthoate(DHNA)-recombinant protein adduct, as measured by an ion exchangechromatography (IEC) assay at 310 nm.
 2. The method of claim 1, whereinsaid analytical assay is by HPLC, RP HPLC, HIC HPLC, NMR, massspectrometry, or UV spectroscopy.
 3. The method of claim 1, wherein saiddO₂ is maintained at levels greater than 0% continuously throughout theharvest operations of step (b).
 4. The method of claim 3, wherein theharvest operations comprise a homogenization stage.
 5. The method ofclaim 4, wherein said dO₂ is maintained at about 30% to about 75% priorto homogenization.
 6. The method of claim 4, wherein said dO₂ ismaintained at levels greater than 75% prior to homogenization.
 7. Themethod of claim 4 or 5, wherein said dO₂ is maintained at about 50%after homogenization.
 8. The method of claim 4, wherein said dO₂ ismaintained at levels greater than 50% after homogenization.
 9. Themethod of claim 5, wherein said dO₂ is maintained for a period ofgreater than or equal to 1.5 hours.
 10. The method of claim 6, whereinsaid dO₂ is maintained for a period of greater than or equal to 2 hours.11. The method of claim 1, wherein said dO₂ is maintained with overlayor sparged air, with increased back-pressure, or with agitation rate.12. The method of claim 11, wherein the overlay air is from about 0.4vvm to about 0.8 vvm.
 13. The method of claim 12, wherein the overlayair is targeted at 0.6 vvm.
 14. The method of claim 11, wherein theincreased backpressure is between about 1.0 to 30 psi.
 15. The method ofclaim 14, wherein the increase backpressure is targeted at 19 psi. 16.The method of claim 11, wherein the agitation rate is from about 6Watts/L to about 8 Watts/L.
 17. The method of claim 16, wherein theagitation rate is targeted at about 6 Watts/L.
 18. A method of producinga recombinant protein comprising (a) fermenting a menE gene-deletedprokaryotic host cell wherein said prokaryotic host cell has beentransformed with a nucleic acid encoding said recombinant protein, (b)harvesting said recombinant protein; and (c) purifying said recombinantprotein to a FBS, wherein said filtered bulk does not contain detectable1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, asmeasured by an ion exchange chromatography (IEC) assay at 310 nm. 19.The method of claim 18, wherein the recombinant protein yield isincreased by about 20% or greater, by about 30% or greater, by about 40%or greater, by about 50% or greater, by about 60% or greater, ascompared to the yield using a control prokaryotic host cell.
 20. Themethod of claim 1, wherein the fermentation is scale-independent. 21.(canceled)
 22. (canceled)